Currently, infection with HIV is usually diagnosed by the detection of HIV specific antibodies in serum using either ELISA or Western Blot. Prior to development of HIV-specific antibodies, there exists a "window period" of seronegative HIV infection wherein an individual is infected with HIV but is not detected as HIV positive using standard ELISA or Western Blot assay. In one study, this window period has averaged 45 days from the date of infection, with 90% seroconverting within 141 days while 10% of individuals did not seroconvert for up to 6 months (1). Recent improvements in anti-HIV antibody detection systems have reduced the window period of seroconversion to approximately 22 days after infection. However, this estimate included a large 95% CV ranging from 10-34 days (2). One Canadian judicial report stated that "The greatest contributor to residual risk of HIV transmission (by transfusion) is the window period during which donors are infected but have not developed antibodies to a detectable level" (3).
Although every unit of donated blood for transfusion purposes is tested for HIV specific antibodies, the window period puts the safety of the blood supply in jeopardy since a person with seronegative HIV infection could donate a unit of HIV infected blood without being detected. Due to this small, but significant risk, the blood collection facilities screen donors to minimize donations from individuals considered to be at risk for HIV infection. Current estimates of the rate of transfusion related HIV infection from seronegative units of blood vary from 1:36,000 (4) to 1:60,000 (5) to 1:153,000 (6) to 1:250,000 (3). There were a total of 1,1418,916 units of untreated blood components transfused in 1993-1994 in Canada, including plasma, apheresis platelets, single donor platelets, red blood cells and cryoprecipitated AHF (7). Therefore, in Canada alone, the number of HIV infections resulting from transfusions of HIV infected, seronegative blood donations in 1993-1994 could be as low as 6 or as high as 39. Since blood products from individual donations are pooled, a single unit of HIV infected, antibody negative blood could potentially contaminate multiple components. If transfusion infected individuals do not have cause to seek HIV testing, they will spread HIV through intravenous drug use, sexual contacts or maternal-fetal transmission. The numbers of potential infections arising from these individuals during their 10-12 year asymptomatic period of HIV infection cannot be calculated.
The Western Blot and ELISA antibody tests currently in use to screen donated units for the presence of HIV do not detect seronegative HIV infections. Three test kits which are not antibody based are currently commercially available and can detect seronegative HIV infection. Both the quantitative competitive polymerase chain reaction (QC-PCR) marketed by Roche Ltd. and the branch chain DNA (bDNA) marketed by Chiron Corp. detect HIV-specific RNA in plasma of patients infected with HIV. These kits capture HIV RNA from plasma or serum and use different amplification systems to amplify the small amount of RNA present to a measurable signal (8). However, the above two test kit procedures have significant limitations. Both QC-PCR and bDNA cost approximately $100 (USD) per test sample. Neither test kit is currently approved by Health and Welfare Canada for HIV diagnosis in Canada. In two U.S. test sites, bDNA detected only 69% and 75% of HIV antibody positive, asymptomatic patients. (8).
An ELISA test for IRV p24 antigen detects serum p24 antigen and reduces the window period of HIV detection to 18-22 days after infection (2). Although this test has been adopted by the blood industry in both Canada and the United States, the efficacy of this test and its minor reduction in the length of the window period, does not outweigh its financial burden on the public.
Therefore, there remains a need for a reliable, specific and sensitive test of seronegative HIV infection that is affordable and practical on a large scale.
It has been demonstrated that HIV infection of either peripheral blood lymphocytes or the cell line, Jurkat, with HIV, results in enhanced levels of phosphotyrosine containing proteins 4-5 days following infection (9). Levels of phosphotyrosine on two proteins (pp95 and pp55) were increased 30 minutes after exposure of Jurkat to Jurkat transfected with gp120 (a model of syncytia formation, cells were not infected with HIV). Levels of phosphotyrosine containing proteins declined 4 hours after exposure.
It has been reported that of asymptomatic HIV infected patients (average CD4=295 cells/.mu.l), 7/25 had elevated fyn protein and decreased lck protein. In addition, there was no change demonstrated in levels of phosphotyrosine in the resting T lymphocytes from patients infected with HIV (9).
Juszczak et al have demonstrated that gp120 and gp120-derived peptides transiently induced tyrosine phosphorylation and activation of p56.sup.lck in normal, resting peripheral blood lymphocytes and a T lymphocyte cell line, HUT 78 (12). Levels of phosphotyrosine and lck activity rose at 5 minutes, peaked at 15 minutes and returned to control levels 30 minutes following treatment.
Horak et al have demonstrated that treatment of activated T lymphocyte clones with either gp120 or HIV did not alter levels of phosphotyrosine containing proteins or activity of p56.sup.lck when treated for 0.5-15 min.(13).
Hivroz et al have demonstrated that gp120 and gp120-derived peptides transiently induce tyrosine phosophorylation and activation of lck in normal, resting peripheral blood lymphocytes and in a T lymphocyte cell line HUT 78 (14). Levels of phosophotyrosine and lck activity rise at 5 minutes, peak at 15 minutes and return to control levels 30 minutes after treatment with either gp120 or gp120-derived peptides.
Kaufmann et al demonstrated that treating resting peripheral blood lymphocytes with gp 120 for 1 hour did not induce a change in levels of phosphotyrosine containing proteins, intracellular calcium, protein kinase C (a serine/threonine protein kinase) or arachidonic acid metabolites (15).
Orloff et al have demonstrated that infecting normal, resting peripheral blood lymphocytes and activated lymphocyte blasts with HIV does not alter levels of phosphotyrosine or activity of p56.sup.lck for 1-120 minutes after infection (16).
Phipps et al have demonstrated that gp120-derived peptides transiently reduce the activity of lck but transiently enhance the activity of fyn and src in activated peripheral blood lymphocyte blasts (17).
Current kinase assays present significant problems. Standard in vitro immune complex kinase assays are not amenable to the screening of numerous samples due to their complex, multi-step procedure and are only semi-quantitative using densitometric analysis of autoradiographs. They also suffer from the same two drawbacks as existing quantitative kinase assays.
The UBI quantitative PTK (protein tyrosine kinase) assay kit currently available relies on immunoprecipitated PTK-mediated tyrosine phosophorylation of synthetic amino acid peptide substrates. Immunoprecipitated PTK is incubated with the peptide and .gamma..sup.32 P-ATP, the reaction stopped and an aliquot of kinase mixture transferred to filter discs, washed, dried and the degree of phosphorylation determined by liquid scintillation counting. The kit can distinguish between different families of PTK but not between different members of the same family. The Pierce quantitative PTK or PKC ELISA-based calorimetric assay binds a biotinylated tyrosine-containing peptide to avidin-treated 8-well microtitre strips. PTK-containing immunoprecipitates are added to phosphorylate the peptides, residual proteins washed out, HRP-labelled anti-phosphotyrosine added, residual antibody washed out, HRP substrate added and the resulting color that is generated is quantified against a standard phosphotyrosine-containing peptide, spectrophotometrically. The kit cannot distinguish between different PTK.
The two major disadvantages of existing kinase assays are:
1) the need to extensively process blood in order to isolate a target cell population; and PA1 2) the lack of standardization of immunoprecipitation. PA1 (a) (i) exposing said immune complex to a mixture of ATP and labelled anti-phosphotyrosine antibody, wherein said label is chosen from an enzyme, fluorescent label, radioactive label and an avidin/biotin system employing any of these labels, so as to achieve tyrosine phosphorylation of said immune complex and said blocking agent; PA1 (b) (i) exposing said immune complex to a mixture of tyrosine phosphorylated polypeptide and malachite green so as to achieve cleavage of phosphate from said phosphorylated tyrosine to provide free phosphate; PA1 (c) (i) exposing said immune complex to labelled antibodies specific for said bound enzyme in said immune complex, wherein said label is chosen from an enzyme, fluorescent label, radioactive label and an avidin/biotin system employing any of these labels, so as to achieve binding of the labelled antibody to the antigen;
1) Current density gradient-based techniques for the isolation of peripheral blood mononuclear cells PBMC from whole blood require a) the extensive handling of biohazardous material; b) the need to transfer samples to new processing tubes at least three times prior to immunoprecipitation, which increases the risk of mixing up samples; c) the extensive washing of cells to remove the residual density gradient media and risking the loss of the sample; d) the enhanced cost of density gradient medium and blood processing. PA2 2) Investigators currently conjugate anti-PTK to protein A-sepharose (or similar solid matrix material) to prepare immune conjugates used to immunoprecipitate PTK from cell lysates. Protein A-sepharose is a slurry of microscopic beads that require washing of both immunoconjugates and immunoprecipitates prior to processing. Washing and aspiration of waste supernatants enhances processing time and increases the risk of loss of sample which can result in the application of different amounts of immunoprecipitate to the kinase reaction enhancing inter-test variability. PA2 (ii) binding of the labelled anti-phosphotyrosine to the phosporylated tyrosine; and PA2 (iii) measuring the amount of bound anti-phosphotyrosine antibody, PA2 (ii) reacting said free phosphate with malachite green so as to achieve a colour change; and PA2 (iii) measuring said colour change on a spectrophotometer; and PA2 (ii) measuring the amount of bound labelled antibody,